The 2,4,6 isomer of trinitrotoulene (TNT) and its environmental transformation products are the most important munitions-derived pollutants encountered at military installations. Large amounts of these compounds have been released into the environment during manufacturing and disarming of ordnance. Remediation of sites contaminated with explosives is required by current statutes, and cleanup criteria have often been set on the order of 25 mg/kg for TNT. It is estimated that the United States probably has in excess of a billion tons of soil contaminated by TNT at &gt;25 mg/Kg. Incineration of soils costs on the order of $1,200/cubic yard. Biological methods for cleanup of this pollutant would be cheaper but have been slow in the making because of the difficulty microbial systems have in metabolizing TNT. Metabolism of aromatic compounds requires cleavage of the ring system, and the chemical structure of TNT makes this difficult. It is postulated that the electrophilic nature and the orientation of the nitro groups prevents biological mineralization. Rieger and Knackmuss, "Basic Knowledge and Perspectives on Biodegradation of 2,4,6,-Trinitrotoulene and Related Nitroaromatic Compounds in Contaminated Soil". in: Bioremediation of Nitroaromatic Compounds. Edited by J.. C. Spain. #49 in the series Environmental Science Research. (Plenum Press, New York, N.Y., 1995), report that oxygenase reactions are "unknown" for trinitro compounds due to the electron withdrawing power of the three nitro groups. The electron withdrawing effect of the nitro groups makes the ring electron deficient and makes electrophilic attack by hydroxylase enzymes difficult. Inhibition of hydroxylation of the ring effectively eliminates TNT as a microbial nutrient and explains the remarkable persistence of this environmental pollutant.
Although the electron withdrawing effect of the three nitro groups may be sufficient in itself to protect TNT from microbiological attack, it is believed that the orientation of the nitro groups is the primary factor in this resistance. During synthesis of TNT, the initial nitration of the toluene ring occurs preferentially at the ring positions ortho or para to the methyl substituent. Additional nitrations of the ring become more difficult because of the "deactivation" of the ring caused by the cumulative electron-withdrawing effect of each nitro group. An additional result of this deactivation is that new nitro groups are added ortho and para to the methyl group and meta to each other. ##STR1## The resultant meta spacing of the three nitro groups ensures that any additional substituents added to the ring will also be selectively positioned meta to each other. However, biological cleavage of aromatic rings requires emplacement of phenolic substituents ortho or para to each other, Dagley, S. "Microbial Degradation of Organic Compounds in the Biosphere." American Scientist, 63:681-689 (1975); Simpson and Evans "The Metabolism of Nitrophenols by Certain Bacteria." The Biochemical Journal, 55:24 (1953). Thus, even if hydroxylations of the aromatic ring were to occur, the required spacing of the phenolic hydroxyl groups could not be achieved without subsequent isomerization. It is likely that the meta orientation of TNT and its resistance to hydroxylation results in inhibition of biological cleavage and these factors are responsible for the persistency of TNT as a contaminant in natural environments.
On the other hand, TNT can be biologically mineralized under laboratory conditions. The probable reaction sequence begins with reduction of two or more of the nitro groups to primary amines. In subsequent steps, the amino groups undergo transamination reactions that yield a ring structure with meta substituted phenolic substituents. In later steps, the methyl carbon is removed and rearrangements occur that move the phenolic groups to an ortho configuration. The ring is then oxidatively cleaved. See Selivansvskaya et al. "Terminal steps in Prepatory Metabolism of 2,4,6,-Trinitrotoulene in Pseudomonas flourescens." Mikrobiologia, 56:6, 1040-1041 (1986) as cited in Walsh, Environmental Transformation Products of Nitroaromatics and Nitramines. US Army Corps of Engineers Cold Regions Reseach & Engineering Laboratory, Special Report 90-2, ##STR2## Although mineralization of the ring carbons of TNT has been demonstrated in the laboratory, it appears that TNT is not utilized as a nutrient by microorganisms in natural environments. This most likely results from the fact that the required sequence of reduction, transamination, isomerization, and oxidation is disfavored if other food sources are available.
It follows from the above that conversion of TNT to a compound that contains only two nitro groups and "unsubstituted" ring carbons in the ortho or para configuration would greatly enhance its potential for microbial degradation. In addition, if the compound that was formed already had the two phenol substituents in place and properly orientated, metabolism of TNT would be accelerated. ##STR3##
The present invention relates to converting TNT to a mixture of dinitro compounds that contain two or three hydroxy substituents and then degrading these reaction products with microbial systems.